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CASE FILES: BIOCHEMISTRY
HSV can infect many cell types (including macrophages, lymphocytes, and
neurons) causing lytic, persistent, and latent infections. Because of nerve cell
involvement in latent infections, a recurrence of the disease is often preceded
by a prodrome (sensations such as burning or tingling). Initial HSV-1 and
HSV-2 infections are usually established on mucous membranes. While HSV-1
and HSV-2 are respectively referred to as oral-herpes and genital-herpes based
on their typical site of infection, HSV-1 and HSV-2 can be found on both oral
and genital tissue.
Diagnosis can be made by examining infected tissue or cells for character-
istic cytopathologic effects, virus isolation and culture, or serology. Molecular
methods (such as, DNA
in situ
hybridization, and PCR of vesicle fluid or
scrapings) are also used for diagnosis. The molecular methods are gaining
favor because they yield rapid results and identify the viral type or strain.
Because of these diagnostic advantages, PCR is the preferred method for diag-
nosis of HSV encephalitis.
The polymerase chain reaction (PCR) is an
in vitro
method for the expo-
nential amplification of a specific DNA region. Today, PCR is one of the
most important biochemical techniques and is applied in virtually all fields of
modern molecular biology. The ingredients for PCR reactions are very simple:
the target DNA to be amplified, a heat-stable DNA polymerase, the four
deoxyribonucleotides, two oligonucleotide primers, and a reaction buffer.
The entire PCR reaction is carried out in a single tube containing all these nec-
essary components. The target DNA segment is amplified with a high degree
of specificity by using two DNA oligonucleotide primers that are comple-
mentary to sequences on the 3'-flanking regions of opposite strands of the tar-
get segment. The PCR amplification occurs by repeated cycles of three
temperature-dependent steps called denaturation, annealing, and elonga-
tion as schematically presented in Figure 6-1. Native DNA exists as a double
helix; therefore the first step, denaturation, of the process separates the two
DNA chains by heating the reaction mixture to 90°C to 95°C (194°F to
203°F). In the second step, annealing, the reaction mixture is cooled to 45°C
to 60°C (113°F to 140°F) so that the oligonucleotide primers can bind or
anneal to the separated strands of the target DNA. In the final step of the
process, elongation, the DNA polymerase adds nucleotides to the 3' ends of
the primers to complete a copy of the target DNA template. A heat-stable
DNA polymerase obtained from the thermophilic bacterium
T. aquaticus
is
used to synthesize the new strands in most PCR reactions. Since this Taq DNA
polymerase works the best at around 72°C (161.6°F), the temperature of the
PCR mixture is raised to that temperature for elongation to proceed efficiently.
At the end of a three-step cycle, each target region of DNA in the vial has been
duplicated. This three-step cycle is then repeated multiple times. Each new
DNA strand can then act as a new template in the next cycle, yielding approx-
imately one million copies of a target DNA after 20 cycles. Therefore PCR
is a method able to amplify any DNA sequence virtually without limit and
allows the separation of the nucleic acid of interest from its context.
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